Microorganisms are used for the production of various useful substances such as the production of biofuels. Microbial culture for the production of biofuels and substances is generally premised on the pure culture of a microorganism of interest (hereinafter referred to as “target microorganism”). This requires the sterilization treatment (also referred to as “disinfection treatment”. Unless otherwise noted, the words “sterilization” and “disinfection” are treated as synonymous with each other) of culture media and instruments and the use of various drugs such as antibiotic substances for the prevention of contamination with microorganisms other than the target microorganism. In a case where an antibiotic substance is used, pure culture is maintained by introducing an antibiotic-resistance gene into the target microorganism and culturing the target microorganism in a sterilized culture medium containing an antibiotic substance corresponding to the antibiotic-resistance gene. It has long been known that genes resistant to drugs such as ampicillin and tetracycline are used as selective markers (e.g. see Non-patent Literature 1.).
However, a lager culture scale leads to higher costs of input energy for sterilization of culture media and the like and higher costs of equipment for sterilization. Further, a larger culture scale also leads to a problem of higher cost for procuring drugs, such as antibiotic substances, that are added to the culture media. Furthermore, since a waste liquid containing an antibiotic substance fosters the emergence of antibiotic-resistant bacteria, the treatment of a waste liquid containing an antibiotic substance must be strictly performed. This makes it also necessary to take the trouble and cost to treat a waste liquid containing an antibiotic substance.
Further, a conventional culturing method that uses an antibiotic substance may cause a microorganism having an antibiotic-resistance gene to decompose an antibiotic substance contained in a culture medium. It is known that the subculture of such a microorganism having an antibiotic-resistance leads to a decrease in the amount of an antibiotic substance contained in a culture medium and ends up in failure to select the target microorganism.
Meanwhile, as a matter of course, the adoption of an economical culturing method, such as culturing in an open system for simplification of equipment or reducing the number of process steps such as sterilization for cost reduction in a culturing step, makes it impossible to industrially purely culture the target microorganism.
Therefore, the foregoing problem is a major obstacle to the practicability of the production of a substance using a microorganism.
Incidentally, Non-patent Literature 2 discloses isolating a phosphite-oxidation gene or a hypophosphite-oxidation gene from Pseudomonas stutzeri by making a cosmid clone of the P. stutzeri genome and isolating the gene by using as an index the acquisition by a host cell (Pseudomonas aeruginosa) of the capability to utilize phosphorous acid or hypophosphorous acid. However, Non-patent Literature 2, too, does not disclose using a phosphite dehydrogenase gene as a selective marker or selectively culturing the target microorganism while inhibiting the growth of microorganisms other than the target microorganism. It should be noted in Non-patent Literature 2, too, culture is of course performed under sterile conditions. Further, Non-patent Literature 2 makes no mention of a culture medium free of an antibiotic substance, either. Furthermore, Non-patent Literature 2, which discloses a technology for isolating a phosphite dehydrogenase gene, is different in technological thought from the present invention.